This invention relates to luminescent reagents.
Specific binding assays provide an economical means for detecting and measuring an analyte present in low concentrations in a sample. Specific binding assays are based upon the interaction of two bindable substances, one the analyte and the other a specific binding partner, which specifically recognize each other. Examples of specific binding partners whose interaction can serve as the basis for a specific binding assay include antigens-antibodies, biotin-avidin, nucleic acid probes, enzymes-substrates, enzymes-inhibitors, enzymes-cofactors, chelators-chelates, and cell surface receptor pairs. Assays involving other specifically bindable substances are also known and within the scope of the present invention. Specific binding assays have shown great utility in determining various analytes in biological, medical, environmental, agriculture and industrial applications.
A variety of assays using the principles of the specific binding approach are known, and several have become important diagnostic tools. In one such type of specific binding assay, the immunoassay, the analyte is an antibody, antigen, or hapten, and is made to react with another member of this group. While the background discussion will focus on such immunoassays, this focus is made for clarity of presentation, and is not to be interpreted as limiting of the invention.
A variety of labeling reactions have been proposed for use in specific binding assays, including radioactive, enzymatic, chromogenic and luminogenic procedures. In a radioactive labeling procedure, the component conjugated with the specific binding partner is an atom or molecule which emits radioactivity. Chromogenic and luminogenic labeling reactions are chemically more complex, in that several reactants may be involved. The chromophore or lumiphore may itself be the label in the reaction, or a catalyst, typically an enzyme, may be used as the label. When the catalyst is used as the label, it will react with catalytic substrates which in turn produce color or luminescence. The remaining components of the reaction, that is, those not conjugated to the binding partner, are supplied in a chromogenic or luminogenic reagent medium, so that the uniting of the labeled conjugate and the reagent medium results in the desired color change or light emission, respectively.
Luminescent labels are attractive alternatives for use in specific binding assays for a variety of reasons. Luminescence is broadly defined as the production of visible light by atoms that have been excited by the energy produced in a chemical reaction, usually without an associated production of heat. Chemical energy excites electrons in the light-emitting molecules to higher energy states, from which electrons eventually fall to lower energy states with the emission of quanta of energy in the form of visible light. Luminescence is observed in several synthetic chemical compounds and also in naturally occurring biological compounds such as found in fireflies and certain varieties of fish.
One of the most important families of chemiluminescent molecules are the phthalylhydrazides. The most familiar member of this family is luminol, or 5-amino-2,3-dihydro- 1,4-phthalazinedione, which has a gross chemical composition of C.sub.8 H.sub.7 N.sub.3 O.sub.2 and a double ring structure with a melting point of about 320.degree. C. Luminol is commercially available from several suppliers and is well characterized. Certain luminol analogs are also chemiluminescent, such as those wherein the position of the amino group is shifted (e.g., isoluminol, the amino group being at the 6 position), or is replaced by other substituents, as well as annelated derivatives and those with substitution in the nonheterocyclic ring. Some luminol analogs produce light more efficiently than does luminol itself, while others have lower efficiency. (As used herein, the term "luminol" encompasses such related species.)
Generally, luminol produces light in an oxidizing reaction, wherein the luminol combines with oxygen or an oxidizer to produce a reaction product and photons at a wavelength of about 425-450 nanometers (nm). The precise reaction formula and the quantum efficiency of light production, i.e., the ratio of luminescing molecules to total molecules of the luminescent species, depend upon the medium in which the luminol resides, temperature and other reaction conditions. Typical oxidizers used in conjunction with luminol include oxygen, hydrogen peroxide, hypochlorite, iodine and permanganate.
The oxidation of luminol with the associated production of light occurs rather slowly at ambient temperatures, unless the reaction is catalyzed. A variety of different substances can catalyze the reaction, including organic enzymes, e.g., horseradish peroxidase, other organic molecules such as microperoxidase and heme, positive metallic ions such as the cupric ion, and negative ions such as the ferricyanate ion.
Luminescent molecules would appear to be highly desirable as tags in specific binding assays because of their stability, sensitivity, the potential ease of detecting their emitted visible light and their lack of toxicity. Commercial luminol, however, has proven to be unsuitable for such purposes. There exists a need for specific improvements in the light emission characteristics of the reaction for use with such assays. Heretofore, commercial luminol has not shown sufficient activity to be useful to measure analytes at low concentrations in specific binding assays. The light emission intensity of the luminol reaction may be sufficient where high concentrations of catalyst are employed and where highly sophisticated and sensitive photometers are available, but the luminescent intensity has not been sufficient with low concentrations of catalyst and where other detection media such as photographic film or less sensitive photometers are used.
While the luminol reaction therefore offers important potential benefits in the measurement of the presence and amount of a reaction component, for many potential applications, the intensity of the emitted light is too low. Further, the light emitted from commercial luminol exhibits an early flash of light within the first few seconds of the initiation of the reaction, followed by a progressive and rapid decrease in light emission over time. The integrated light intensity during any fixed period of time is therefore likely to be different from that measured over any other equal period of time. This variability may result in irreproducibility between tests. Desirably, there would be some period of time during which the light emission from the luminol reaction is relatively constant, so that the measurement of integrated light intensity could begin at different times after initiation of the reaction, but within the period of constant light output, without variability of the results. This would eliminate the requirement that the reagents be added to a solution fixed in front of the luminescence detector which puts severe constraints on the light measuring system.
Higgins et al, U.S. Pat. No. 4,743,541, disclose that the intensity and duration of emitted light from luminol can be considerably improved by repeatedly dissolving and recrystallizing the luminol until sulphide and hydrazine levels are below about 100 ppm.
The production of chemiluminescence with luminol comprises dissolving the luminol in an organic solvent, such as DMSO or acetone, or in a strong base and diluting the solution in a buffer of desired pH. The amount of luminol that can be dissolved is severely limited by the relative insolubility of luminol in water at a pH below 10.
When luminol is covalently attached to carriers such as protein, its chemiluminescence is quenched. Isoluminol, although less efficient in light production than luminol, is quenched to a lesser degree by covalent attachment. The noncovalent attachment of luminol to bovine serum albumin prevents quenching and solubility problems, but "leaks" luminol into the solution by forming an equilibrium between bound and unbound luminol, thus decreasing the specificity of luminol/carrier dependent immunoassays and enzyme-linked assays.
There is a need for a luminescent probe which is water soluble, is highly quantum efficient, and provides long-lived chemiluminescence.
U.S. Pat. No. 5,003,050, issued Mar. 26, 1991, to Johnathan L. Kiel and Gerald J. O'Brien, discloses a water-soluble luminescent compound having repeating units of the formula: ##STR1## The product, which is a polymer having repeating units comprising diazo-linked luminol and hydroxyindole, is referred to as diazoluminomelanin (DALM), since one of the precursors to this product, 3-amino-L-tyrosine (3AT) is closely related to the biological substrates which are converted into melanin.
DALM is water soluble, having an apparent pKa for solubility about pH 5.0. DALM does not require a catalyst for chemiluminescence. The duration of the reaction is in excess of 52 hours. In contrast, luminol requires a catalyst; with microperoxidase as the catalyst, luminol has shown peak luminescence at 1 sec and half-lives of light emission of 0.5 and 4.5 sec at pH 8.6 and 12.6, respectively. The chemiluminescence yield of DALM is better at pH 7.4 than at pH 9.5, although it still provides a strong signal at strongly basic pHs. DALM also produces chemiluminescence at pH 6.5 which is about the same intensity as that produced at pH 9.5.
DALM can be used for chemiluminescent immunoassays for biological and chemical agents; in radiofrequency and ionizing radiation dosimeters; and for RNA/DNA hybridization assays for viruses and genetic detection.
Also disclosed in U.S. Pat. No. 5,003,050 is a method for preparing DALM which comprises reacting 3AT with an alkali metal nitrite, and reacting the resulting diazonium salt with luminol. The method involves the use of organic solvents such as dimethylsulfoxide and acetone. On a large scale, handling and disposal of such solvents could be both dangerous and difficult.
We have discovered that a particular strain of Escherichia coli bacteria containing a plasmid constructed with a barley nitrate reductase gene fragment, grown on a nitrate/luminol/3-amino-L-tyrosine medium produces large quantities of DALM. Additionally, the E. coli containing the recombinant DNA plasmid can be employed for the purpose of detecting physical, chemical and/or radiation stressors.
Accordingly, it is an object of the present invention to provide an improved biosynthetic method for producing DALM.
Another object of the present invention is to provide methods for detecting physical, chemical and/or radiation stressors.
Other objects, aspects and advantages of the present invention will be apparent to those skilled in the art from a reading of the following detailed disclosure of the invention.